Coenzyme nicotinamide adenine dinucleotide (NAD, including the oxidized form-- NAD+ and reduced form--NADH) and the phosphorylated form--nicotinamide adenine dinucleotide phosphate (NADP, including NADP+ and NADPH) are two of the most important biological electron carriers. Most NAD(P) dependent redox enzymes show a preference of either NADP or NAD as an electron acceptor or donor depending on their unique metabolic roles. In biocatalysis, the low enzymatic activities with unnatural coenzymes have made it difficult to replace costly NADP with economically advantageous NAD or other biomimetic coenzyme for catalysis. This is a significant challenge that must be addressed should in vitro biocatalysis be a viable option for the practical production of low-value biocommodities (i.e., biohydrogen). There is a significant need to first address the coenzyme selectivity of the NADP-dependent dehydrogenases and evolve mutated enzymes that accept biomimetic coenzymes. This is a major focus of this dissertation. Establishment of efficient screening methods to identify beneficial mutants from an enzymatic library is the most challenging task of coenzyme engineering of dehydrogenases. To fine tune the coenzyme preference of dehydrogenases to allow economical hydrogen production, we developed a double-layer Petri-dish based screening method to identify positive mutant of the Moorella thermoacetica 6PGDH (Moth6PGDH) with a more than 4,278-fold reversal of coenzyme selectivity from NADP+ to NAD+. This method was also used to screen the thermostable mutant of a highly active glucose 6-phosphate dehydrogenase from the mesophilic host Zymomonas mobilis. The resulting best mutant Mut 4-1 showed a more than 124-fold improvement of half-life times at 60oC without compromising the specific activity. The screening method was further upgraded for the coenzyme engineering of Thermotaga maritima 6PGDH (Tm6PGDH) on the biomimetic coenzyme NMN+. Through six-rounds of directed evolution and screening, the best mutant showed a more than 50-fold improvement in catalytic efficiency on NMN+ and a more than 6-fold increased hydrogen productivity rate from 6-phosphogluconate and NMN+ compared to those of wild-type enzyme. Together, these results demonstrated the effectiveness of screening methods developed in this research for coenzyme engineering of NAD(P) dependent dehydrogenase and efficient use of the less costly coenzyme in ivSB based hydrogen production. / Ph. D.

Ncd is a member of the kinesin family of motor proteins. Ncd is involved in the processes of meiosis and early mitosis in <i>D. melanogaster</i>. PCR-mediated site-directed mutagenesis was utilized to introduce specific mutations into pET/MC6, a construct containing the motor domain of ncd. Six mutations were generated, two at glutamic acid residue 656, two at proline residue 649, one at arginine residue 623, and one double mutant at arginine residue 623 and threonine residue 632. Mutants proteins were expressed in bacteria and further characterized. Mutagenesis of the proline or glutamic acid residues resulted in insoluble proteins. The one exception is the mutagenesis of glutamic acid residue 656 into a glutamine, which resulted in a partially soluble protein. Mutagenesis of the arginine residue into an alanine (MC6-A623) resulted in a soluble protein while the double mutation of the arginine and threonine was insoluble. MC6-A623 exhibited a similar S-sepharose ion exchange chromatography binding and elution profile as MC6. Peptide antibodies made to conserved ncd motor domain sequences also recognized MC6- A623. The affinity of MC6-A623 (under the conditions tested) for microtubules was less than MC6. Most interestingly, under the conditions tested, MC6-A623 did not exhibit an increased ATPase rate in the presence of microtubules, a hallmark of the kinesin family of microtubule motor proteins. Analysis of the published ncd crystal structure, other motor protein sequences, and the experimental results of the mutagenesis of arginine residue 623, suggest that this residue is involved in the binding of MC6 to microtubules. / Master of Science

Thesis advisor: Abhishek Chatterjee / Thesis advisor: Jia Niu / Directed Evolution has emerged as an invaluable tool for advancing protein functions in both research and industry. Our lab has pioneered a directed evolution platform in mammalian cells, utilizing an AAV delivery vector to package a DNA library and linking the biomolecule of interest to AAV production. During my tenure in Prof. Chatterjee's lab, I focused on harnessing our lab’s directed evolution platform, known as Virus-Assisted Directed Evolution of tRNA (VADER), to develop highly efficient tRNAs for genetic code expansion. Additionally, I contributed to the development of the AAV-based selection platform, termed Virus-Assisted Mutational Profiling (VAMP), as a profiling tool. Through the utilization of VAMP, I conducted comprehensive profiling of tRNA and RNA polymerase III promoter sequences. This enabled me to gain insights into regions of flexibility and evolution, ultimately leading to the construction of improved constructs with enhanced activity relative to the starting sequence. / Thesis (PhD) — Boston College, 2024. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

From 1959 -1964, the Prince Edward County, VA School Board closed down its public schools to circumvent the 1954 Brown v. Board of Education Supreme Court ruling declaring separate public schools for Black and White students "inherently unequal" and the 1955 Brown II ruling to desegregate public schools with "all deliberate speed." For five years, more than 1700 African American children received no public education in the county, as White children attended a newly-constructed and private Prince Edward Academy. While some students left Prince Edward to reside with relatives, others were placed with families by the American Friends Service Committee. However, the majority of Black children remained in the county without formalized public instruction. This study investigated the perceived impact of The Closing on adult self-directed learning, lifelong learning, occupational choices and success within a family with sixteen of its twenty-one children forced from school. Via audio-/video-taped interviews, three participants reflected upon their "lived experiences" during and since The Closing. Transcribed data were coded and analyzed based upon the major and underlying research questions guiding the study. Nine major conclusions were drawn from its findings: (a) The Closing perceivably impacted immediate educational goals of participants differently, (b) The Closing perceivably impacted specific and general long-range educational goals, (c) Participants have pursued educational goals via supportive spouses/family members and adult self-directed/lifelong learning measures, (d) Following the re-opening of schools, all respondents graduated high school, and two later enrolled in academic learning centers, (e) Self-directed learning has played an essential role in the lives of all participants, (f) All participants considered themselves life-long learners, (g) The Closing perceivably impacted the career plans of one participant, (h) Respondents acquired manufacturing and/or labor positions and were successfully employed throughout their adult lives, (i) Literacy assistance from family members, self-directed learning, on-the-job training and formalized coursework were perceived as having had a positive bearing on occupational success. The implications of this study suggested resiliency, family dynamics, family values, and narratological significance. Study participants, driven to live productive and successful lives, appeared to have emulated Adult Learning Theory tenets of self-directed, lifelong quests for formally-delivered and informally-acquired knowledge. Recommendations emerging from this study included investigations of School Closing survivors' motivations for adult learning, the role of faith in Closing survivors' lives, The Closing's perceived impact on the Next Generation, ancestral discourse, male birth order relationships, 1951 strikers' guilt, education vs. vocation and growth under adversity. / Ph. D.

Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen that is a substantial threat, particularly in hospital settings, causing severe infections in immunocompromised patients that may lead to death. Pseudomonas aeruginosa harbors a multitude of virulence factors that enable this pathogen to establish both acute and chronic infections in humans. A key determinant of acute infections is a hollow molecular needle structure used for injecting toxins into a host cell, called the type three secretion system (T3SS). The secretion machinery itself is highly complex and, together with the specific secreted factors, requires expression of more than 30 genes. Due to the high energy cost of its synthesis to the organism this system is highly regulated to finely time gene expression to coincide with host contact. ExsA, a member of the AraC-type transcription factor family, is the main transcriptional activator of all the genes necessary for expression of the T3SS. Members of the AraC family are characterized by the presence of two helix-turn-helix (HTH) motifs, which bind to the promoter DNA and activate transcription. ExsA uses its HTH containing C-terminal domain (CTD) to regulate gene expression from 10 different promoters. The N-terminal domain (NTD) of ExsA mediates dimerization and regulation of ExsA-activity. While most AraC-type activators are regulated by a small molecule ligands, ExsA is regulated by another protein, ExsD. As part of a four-protein signaling cascade, ExsD interacts directly with ExsA to prevent transcription of T3SS-associated genes under non-inducing conditions prior to host cell contact. The entire regulatory cascade includes of two additional proteins, ExsC and ExsE. ExsA, ExsC, ExsD, and ExsE follow a partner-switching mechanism to link expression of the secretion system with host cell contact. Our laboratory is working to understand this unique signaling mechanism by determining the molecular basis for the regulation of this important virulence factor. Previous studies in the laboratory have solved the structures of ExsE, ExsC and ExsD, and shed light on how these proteins interact and compete for overlapping binding sites. However, it is still unclear as to how the ExsA and ExsD interact and thus how regulation is mediated at the molecular level. In the presented study, we sought to map the molecular interface between ExsA and ExsD. First, the crystal structure of ExsA-NTD is presented wherein the dimerization interface of the protein was identified. Two of the well-studied AraC-type proteins, AraC and ToxT crystal structures have been solved by others in the presence of their respective ligands. Residues that were involved in ligand binding in AraC and ToxT were aligned with the residues in ExsA and analyzed for interaction with ExsD. However, this canonical binding pocket appeared to be not involved in the interaction between ExsA and ExsD. Structure directed site-specific mutagenesis was carried out to construct many different variants of ExsD and ExsA. Thus constructed variants were purified and analyzed in a functional assay. Using this approach, we were able to identify regions on ExsD and ExsA that are crucial for the interaction and for the regulation of ExsA-dependent transcription. It turns out that backbone interactions between the amino-terminal residues of ExsD and the beta-barrel region of the ExsA-NTD are pivotal. This result explains how ExsA and ExsC compete for ExsD binding, since both target the same regions on ExsD. / PHD

No / Smart mobile devices, which are hand-held electronic devices with an advanced operating system (such as the Android platform) connected via a wireless protocol, have become an integral and essential part of our everyday life, and support both social and workplace activities. However, adopting mobile technology within the workplace setting can give rise to challenges that impact user behaviour and performance. A study was carried out amongst 90 participants located in two countries, using internet connectivity as a case study. Confidence and frustration have previously been connected with technology competence, but this was not applied to a workplace scenario during problem-solving, when users are assigned an unfamiliar smart mobile device. This research focuses on identifying the link between workplace users' levels of confidence and frustration when seeking to independently solve problems whilst completing familiar tasks on new smart mobile devices. A detailed video analysis of users' attitudes and behaviour during problem-solving was conducted, emphasising a correlation between attitudes and behaviour towards completing a task.

Francisella tularensis is a Gram-negative, facultative intracellular coccobacillus and the etiologic agent of tularemia for a wide variety of vertebrate and invertebrate animal species. Several species and subspecies of Francisella are currently recognized. However, the majority of infections are caused by F. tularensis subspecies tularensis (type A) and subspecies holarctica (type B). Given the low infectious dose, multiple transmission routes, severity of illness, and lack of licensed vaccines, F. tularensis has long been considered a potential biological weapon and is now classified as a category A select agent by the National Institutes of Health and the Centers for Disease Control and Prevention. The investigation of the mechanisms of pathogenesis by F. tularensis type A and B strains is hindered by the difficulty and lack of methods to mutate the putative genes that encode for virulence factors. New genetic tools have been developed that have enabled mutagenesis of F. tularensis type A and type B stains. However, site-specific mutations remain difficult to execute or these methods generate random mutations. In this study a novel method was developed to create site-directed mutations in a putative capsule biosynthesis locus to knock out encapsulation of the attenuated F. tularensis live vaccine strain. Two suicide vectors for mutagenesis of F. tularensis were constructed based on the commercial PCR cloning vector pSC-A. These vectors were created by inserting into the cloning site a kanamycin resistance gene boarded upstream by 1.3 kb of N-terminal DNA and downstream by 1.3 kb of C-terminal DNA that flanks the target gene. Cryotransformation was used to introduce the vectors into F. tularensis. Open reading frame (ORF) FTT0793, which may encode for an ABC transporter involved in capsule export, was initially selected for mutagenesis in order to generate a mutant that was nonencapsulated, but could still synthesize capsule and induce a host immune response. Mutagenesis of this gene was successful. However, phenotypic assays could not confirm that the mutant was nonencapsulated compared to the parent. Therefore, adjacent ORFs FTT0798 and FTT0799, which may encode for a galactosyl transferase and mannosyl transferase, respectively, were also deleted to completely knock out capsule synthesis. The resulting mutant appeared to be nonencapsulated as determined by negative staining transmission electron microscopy. In this study, a plasmid and method for generating allelic exchange mutants is reported, which should be useful for generating additional mutants of F. tularensis for use in clarifing the roles of specific genes. This vector is currently being used to make a nonencapsulated mutant of a virulent type A strain to determine the role of capsule in virulence. / Master of Science

The overall goal of this work is to create an open-source digital twin of the wire arc directed energy deposition process using robot operating system 2 for interpass temperature regulation of a maraging steel alloy. This framework takes a novel approach to regulating the interpass temperatures by using in-situational infrared camera data and a closed loop feedback control that is enabled by robot operating system 2. This is the first implementation of robot operating system 2 for wire arc directed energy deposition and this framework outlines a sensor and machine agnostic approach for creating a digital twin of this additive manufacturing process. In-situ control of the welding process is conducted on a maraging steel alloy demonstrating interpass temperature regulation leads to improved as-built surface roughness and more consistent as-built hardness. An evaluation of three distinct weld modes: Pulsed MIG, CMT MIX, and CMT Universal and two primary process parameters: travel speed and wire feed speed was conducted to identify suitable process windows for welding the maraging alloy. Single track welds for each parameter and weld mode combination were produced and evaluated against current weld bead metrics in the literature. Non destructive profilometry and destructive characterization were performed on the single track welds to evaluate geometric features like wetting angle, dilution percentage, and cross sectional area. In addition, the role of material feed rate on heat input and the cross sectional area was examined in relation to the as-built hardness. The robot operating system 2 digital twin provides a visualization environment to monitor and record real time data from a variety of sensors including robot position, weld data, and thermal camera images. Point cloud data is visualized, in real time, to provide insight to the captured weld meta data. Capturing in-situ data from the wire arc directed energy deposition process is critical to establishing an improved understanding of the process for parameter optimization, tool path planning, with both required to build repeatable, quality components. This work presents an open-source method to capture multi-modal data into a shared environment for improved data capture, data sharing, data synchronization, and data visualization. This digital twin provides users enhanced process control capabilities and greater flexibility by utilizing the robot operating system 2 as a middleware to provide interoperability between sensors and machines.

Yes / Dehydroabietic acid (DAA) promotes palladium(0)‐catalysed cyclisations of arene‐tethered 1,7‐enynols and 1,m‐enynoates (m=6,7) to give fused carbocyclic dienes. 6,6,6,5‐Tetracyclic lactones are accessible by one‐pot cycloisomerisation/Diels–Alder reaction/lactonisation from 1,7‐enynols. Furthermore, asymmetric counteranion‐directed catalysis has been developed, which afforded an indene derivative with an all‐carbon quaternary stereogenic center. / NSFC. Grant Number: 21462004 State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources. Grant Number: CMEMR2014A04 2015 GXNSFBA. Grant Number: 139032 GXNU

Thesis advisor: Tim van Opijnen / Although vaccines and antibiotics have been historically successful in combating bacterial infections, limited vaccine coverage and the rise of antibiotic resistance emphasize the need to develop alternative, broadly effective, and/or targeted treatment strategies to reduce the health burden of bacterial infections. Rather than relying on therapeutics solely targeting the bacterial pathogen, such as standard antibiotics, therapies that simultaneously focus on host responses are emerging. In this thesis, we propose 'host-informed therapies' (HITs) in two categories: those that aid patients with fully functional immune systems and those that aid patients with perturbed immune processes, as promising alternative or adjunctive treatment strategies for bacterial infections. The host-pathogen interaction during infection is a highly dynamic process between diverse bacterial pathogens and hosts with varying degrees of susceptibility. Systems biology approaches have provided an understanding of host-pathogen parameters globally through the detection of putative biomarkers for diagnosis and identification of critical interactions to discover novel drug targets. However, there remains a gap in understanding bacterial pathogenesis in the context of designing novel host-informed therapies. Here, we use Streptococcus pneumoniae, the gram-positive pathogen responsible for the majority of bacterial respiratory tract infections worldwide, as a case study to: (1) Generate a genome-wide map of bacterial immune (complement) evasion targets to design novel host-informed therapies, (2) generate a dual host/pathogen transcriptome map to identify signatures of infection outcome, and (3) validate signatures of bacterial antibiotic tolerance in a mouse lung infection model. Overall, this work exemplifies how systems biology methods can elucidate the intricacies of bacterial pathogenesis but, more importantly, aid in the target identification, validation, and design of antibacterial host-informed therapies. / Thesis (PhD) — Boston College, 2023. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.